DE60202278T2 - ACTIVATION OF NATURAL KILLER CELLS BY ADENOSINE A3 RECEPTOR AGONISTS - Google Patents

Abstract

The present invention is based on the surprising finding that adenosine A3 receptor agonists (A3RAg) are capable of activating natural killer (NK) cells. In particular, methods and pharmaceutical compositions for activating NK cells in an individual comprising administering said individual with an effective amount of one or more A3RAg are provided herein. A further method includes treatment of a disease comprising administering to an individual in need of such treatment NK cells a priori activated with an effective amount of at least one A3RAg. The NK cells may be either autologous cells or cells withdrawn from a donor individual. Further provided are pharmaceutical compositions comprising one or more A3RAg in an amount effective to achieve a therapeutic effect, the therapeutic effect comprising activation of NK cells, the pharmaceutical composition optionally comprising physiologically acceptable additives.

Description

Territory of invention

These The invention relates to the therapeutic use of adenosine A3 Receptor agonist for activation of natural killer cells.

State of technology

• – Nilsson J. und Olsson AG. Atherosclerosis 53: 77–82, (1984);
• – Ward AC, et al. Biochem Biophys Res Commun 224: 10–6 (1994);
• – Wilson NJ, et al. Biochem Biophys Res Commun 244: 475–80 (1998);
• – Giblett ER, et al. Lancet 2: 1067–9 (1972);
• – Priebe T, et al. Cell Immunol 129: 321–8 (1990);
• – Miller JS, et al. J Immunol 162: 7376–82 (1999);
• – Hall TJ, et al. Clin Exp Immunol 54: 493–500 (1983);
• – Trinchieri G. Annu Rev Immunol 13: 251–76 (1995);
• – Link AA, et al. J Immunol 164: 436–42 (2000):
• – Hasko G, et al. FASEB 14: 2065–74 (2000);
• – Hasko G, et al. Eur J Pharmacol 358: 261–8 (1998);

The following is a list of prior art documents believed to be important to the description of the prior art in the field of the invention.

• - Nilsson J. and Olsson AG. Atherosclerosis 53: 77-82, (1984);
• Ward AC, et al. Biochem Biophys Res Commun 224: 10-6 (1994);
• Wilson NJ, et al. Biochem Biophys Res Commun 244: 475-80 (1998);
• Giblett ER, et al. Lancet 2: 1067-9 (1972);
• Priebe T, et al. Cell Immunol. 129: 321-8 (1990);
• Miller JS, et al. J Immunol 162: 7376-82 (1999);
• Hall TJ, et al. Clin Exp Immunol 54: 493-500 (1983);
• Trinchieri G. Annu Rev Immunol 13: 251-76 (1995);
• - Link AA, et al. J Immunol 164: 436-42 (2000):
• - Hasko G, et al. FASEB 14: 2065-74 (2000);
• - Hasko G, et al. Eur J Pharmacol 358: 261-8 (1998);

background the invention

Natural killer cells (NK cells) were first in mice conditioned by their ability to identified specific tumor cell targets to be rapidly lysed. These Cells are a small subgroup of peripheral blood lymphocytes, the 5-16% of the entire lymphocyte population and a specific one Form group of lymphocytes without immunological memory and independent of are adaptive immune system.

NK cells convey a set of features that are important to human health and disease are important. For example, it has been found that NK cells face an important defense of the first line Malignant cells are cells as well as viruses, bacteria and Protozoa are infected. additionally these cells participate in immune regulation, hematopoiesis, Reproduction and neuroendocrine interactions. The find that NK cells causing the production of a number of cytokines led to the assumption that NK cells are just as discrete functional T cells Differentiate subgroups with different effects on adaptive immunity.

Summary the invention

It was found in accordance with the present case that adenosine A3 receptor agonists (A3RAg) natural Killer cells (NK cells) activate and that this activation in the presence of adenosine A3 receptor antagonists (A3RAn) reversed becomes.

Adenosine is a ubiquitous nucleoside that is present in all body cells. It is released from metabolically active or stressed cells and then acts as a regulatory molecule. It binds to cells through specific A1, A _2A , A _2B , and A3 G protein-associated cell surface receptors, thereby acting as a signal transduction molecule by regulating levels of adenyl cyclase and phospholipase C [Linden J. The FASEB J 5: 2668-2676 (1991); Stiles GL Clin. Res. 38: 10-18 (1990)].

According to one First of its aspects, the present invention relates to the use of adenosine A3 receptor agonists (A3RAg) for the production of pharmaceutical Compositions for achieving a therapeutic effect, wherein the therapeutic effect comprises the activation of NK cells.

The term "adenosine A3 receptor agonist" as used herein refers to any molecule capable of binding to the adenosine A3 receptor, thereby fully or partially activating said receptor, examples of such molecules being provided hereinafter. A complete agonist for the present purposes should therefore be understood as an agonist that provides the maximum possible response to be studied by activation of its receptor in the art to the preparation, while a partial agonist is to be understood as an agonist which, however high the concentration used, is unable to provide maximal activation of the receptors.

The "effective Quantity "(or" quantity effective for ") for the present Purposes is determined by such considerations determines how they are known in the field. The amount must be effective be to the activation of NK cells in a detectable and preferably in a therapeutically effective amount. A person who is well-versed in the field will know how the effective amount dependent inter alia on the type and severity of the disease to be treated and the treatment approach.

Of the Term "activation from NK "for the present Purposes per se refers to the activation of the cytotoxic or cytostatic effect of NK cells on foreign or abnormal and unwanted cells or the increase the cytotoxic or cytostatic effect of pre-active (i.e. H. already active) NK cells on the unwanted (foreign or abnormal) Cells as well as on the increase from their other biological functions such as the stimulation of Cytokine production.

The The present invention also relates to a method for activation of NK cells ex vivo by providing an effective Amount of adenosine A3 receptor agonist (A3RAg) to said cells.

Of Further, the present invention relates to the use of A3RAg in an effective amount for the manufacture of a medicament, that for the achievement of a therapeutic effect is suitable, wherein the therapeutic effect the activation of NK cells in said Individual includes.

Of the Term "treatment" for herein used purpose refers to the mitigation of unwanted Symptoms associated with a disease, even without cure the disease, z. The reduction of pain; the prevention the manifestation of such symptoms before they arise; the Slowing down the aggravation of symptoms or progression a disease; reducing the severity or healing of the Illness; the acceleration of the natural or conventional healing process; the improvement of the survival rate or the faster convalescence of the individual suffering from the disease suffering; the prevention of the onset of a disease or a Combination of two or more of the above.

The The invention also relates to the use of A3RAg for the preparation a drug that works for the Treatment of a disease or disease that is the activation of NK cells, said NK cells by contacting NK cells ex vivo with an effective one Amount of A3RAg to be activated. Typically, such includes Method of removing NK cells from the subject (i.e. autologous NK cells), exposing such cells to an effective Amount of at least one A3RAg and introduction of the activated NK cells in the receiver (ex vivo activation). Alternatively, the NK cells may be from a Donors are removed. Such donated NK cells can after Activation with the A3RAg taken in a pretreated donor or after removal in vitro and before administration be activated to the receiving individual. This aspect of Invention is also known by the name "adoptive transfer", according to which one immunity by transferring immunocompetent cells from pretreated cells Donor is transferred to a non-immune recipient.

Of the Term "a priori activation " Activation of NK cells in either a cell or a cell Tissue culture or in an animal model wherein the cells, tissue or the animal is treated with a quantity of A3RAg, which is effective for activating NK cells, and then cells or Tissue preparations containing said activated NK cells therein, from culture or animal for administration to non-immune individual who needs it, removed. The activated NK cells administered to an individual are preferably, although not exclusive, autologous cells.

Of Further, the present invention provides a pharmaceutical composition for the achievement of a therapeutic effect, wherein the composition comprises an A3RAg in an amount that is effective is to achieve said therapeutic effect, wherein the therapeutic Effect includes the activation of NK cells.

Short description of drawings

Around to understand and recognize the invention, as in practice carried out is now a preferred embodiment by way of a not restrictive Example with reference to the accompanying figures, in which:

1 shows the effect of treating an adenosine A3 receptor agonist, CI-IB-MECA, on human peripheral blood NK cells after incubation of the cells with 10 nM CI-IB-MECA, as well as the reversed effect in the presence of an adenosine A3 receptor antagonist ( MRS1220), as determined by the amount of lysis (% lysis) of the target cells (K562 leukemia cells) by the activated NK cells.

2 Figure 12 shows the effect of various doses (10-1000 μg / kg body weight) of CI-IB-MECA administered orally to mice on the activation of murine NK cells by the percentage (of the total release) of the release of ⁵¹ Cr from YAC lymphoma cells labeled with Na ₂ [ ⁵¹ Cr] O ₄ .

3 Figure 12 shows the time-dependent stimulatory effect of CI-IB-MECA on the activity of NK cells after oral administration of CI-IB-MECA (10 μg / kg body weight) to mice for four consecutive days, determined by the percentage (of the total) Release) of the release of ⁵¹ Cr.

4 Figure 12 shows the amount of IL-12 in serum samples derived from CI-IB-MECA treated mice (10 μg / kg body weight).

5A - 5F shows the inhibitory effect of CI-IB-MECA on the development of melanoma and colon carcinoma in mice ( 5A . 5B ) with an increase in the amount of IL-12 in the corresponding melanoma or colon carcinoma-bearing mice (each 5C . 5D ). Also, increased NK cell activity (determined by the percentage (from total release) of ⁵¹ Cr release) in the spleen from CI-IB-MECA-treated melanoma or colon carcinoma-bearing mice compared to untreated mice ( 5E . 5F , respectively).

6 shows the inhibitory effect of grafted CI-IB-MECA "activated" NK cells on tumor cell growth in B16-F10 melanoma-bearing mice, as determined by the number of metastatic pulmonary foci appearing after grafting the "activated" NK cells developed on melanoma-bearing mice.

Detailed description the invention

As will be shown in the following examples, it has been found that CI-IB-MECA, an A3RAg, produces NK cells in a biologically significant Amount activated both in vitro and in vivo. Obviously This find has a therapeutic significance, since NK cells at a Participate in a series of biological processes, including the Defense against malignant and infectious diseases, immune regulation, hematopoiesis, Reproduction and neuroendocrine interactions.

According to one In its aspects, the present invention provides a method Activation of natural killers (NK) cells available ex vivo.

The properties and methods of producing some adenosine A3 receptor agonists are discussed in detail inter alia in US Pat US 5,688,774 ; US 5,773,423 . US 5,573,772 . US 5,443,836 . US 6,048,865 , WO 95/02604, WO 99/20284 and WO 99/06053, WO 97/27173 and other publications available to those skilled in the art.

worin R₁ ein Alkyl-, Hydroxyalkyl-, Carboxyalkyl- oder Cyanoalkyl oder eine Gruppe der folgenden allgemeinen Formel (II) darstellt:

in welcher:
Y ein Sauerstoff, Schwefel oder Kohlenstoffatom ist;
X₁ H, Alkyl, R^aR^bNC(=O)- oder HOR^c- ist, worin
R^a und R^b gleich oder verschieden sein können und aus Wasserstoff, Alkyl, Amino, Halogenalkyl, Aminoalkyl, BOC-aminoalkyl und Cycloalkyl ausgewählt sind oder so miteinander verbunden sind, dass sie einen heterocyclischen Ring bilden, der zwei bis vier Kohlenstoffatome enthält; und
R^c ist aus Alkyl, Amino, Haloalkyl, Aminoalkyl, BOC-Aaminoalkyl und Cycloalkyl ausgewählt;
X₂ ist Wasserstoff, Hydroxyl, Alkylamino, Alkylamido oder Hydroxyalkyl;
X₃ und X₄ sind unabhängig voneinander Wasserstoff, Hydroxyl, Amino, Amido, Azido, Halogen, Alkyl, Alkoxy, Carboxy, Nitrilo, Nitro, Trifluor, Aryl, Alkaryl, Thio, Thioester, Thioether, -OCOPh, -OC(=S)OPh oder X₃ wie auch X₄ sind Sauerstoffe, die mit ≥C=S so verbunden sind, dass sie einen 5-gliedrigen Ring bilden, oder X₂ und X₃ einen Ring der Formel III bilden:

wobei R' und R'' unabhängig voneinander eine Alkylgruppe darstellen;
R₂ ist aus Wasserstoff, Halogen, Alkylether, Amino, Hydrazido, Alkylamino, Alkoxy, Thioalkoxy, Pyridylthio, Alkenyl; Alkinyl, Thio und Alkylthio ausgewählt; und
R₃ ist eine Gruppe der Formel -NR₄R₅ worin
R₄ ein Wasserstoffatom oder eine Gruppe ist, die aus Alkyl, substituiertem Alkyl oder Aryl-NH-C(Z)- ausgewählt ist, wobei Z, O, S oder NR^a ist, wobei R^a die oben genannten Bedeutungen hat; und im Falle, dass R₄ Wasserstoff ist; dann wird
R₅ aus R- und S-1-Phenylethyl-, Benzyl-, Phenylethylgruppen ausgewählt, die in einer oder mehreren Positionen mit einem Substituenten substituiert oder nicht substituiert sind, der aus Alkyl, Amino, Halogen, Haloalkyl, Nitro, Hydroxyl, Acetamido, Alkoxy und Sulfonsäure oder einem Salz davon; Benzodioxanmethyl, Fururyl, L-Propylalanyl-Aminobenzyl, β-Alanylamino-Benzyl, T-BOC-β-Alanylaminobenzyl, Phenylamino, Carbamoyl, Phenoxy oder Cycloalkyl ausgewählt ist; oder R₅ ist eine Gruppe der folgenden Formel:

oder, falls R₄ ein Alkyl oder Aryl-NH-C(Z)- ist, dann ist R₅ aus der Gruppe ausgewählt, die aus Heteroaryl-NR^a-C(Z)-, Heteroaryl-C(Z)-, Alkaryl-NR^a-C(Z)-, Alkaryl-C(Z)-, Aryl-NR-C(Z)- und Aryl-C(Z)- besteht; wobei Z ein Sauerstoff, Schwefel oder Amin ist; oder ein physiologisch verträgliches Salz der oben genannten Verbindung.According to one embodiment of the invention, the A3RAg is a compound of the general formula I:

wherein R _{1 represents} an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or a group of the following general formula (II):

in which:
Y is an oxygen, sulfur or carbon atom;
X _{1 is} H, alkyl, R ^a R ^{b is} NC (= O) - or HOR ^c -, in which
R ^a and R ^{b may be the} same or different and are selected from hydrogen, alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl and cycloalkyl or joined together to form a heterocyclic ring containing from two to four carbon atoms; and
R ^c is selected from alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl and cycloalkyl;
X ₂ is hydrogen, hydroxyl, alkylamino, alkylamido or hydroxyalkyl;
X ₃ and X ₄ are independently hydrogen, hydroxyl, amino, amido, azido, halogen, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, -OCOPh, -OC (= S ) OPh or X ₃ as well as X ₄ are oxygens which are connected to ≥C = S so that they form a 5-membered ring, or X ₂ and X _{3 form} a ring of the formula III:

wherein R 'and R "independently represent an alkyl group;
R ₂ is selected from hydrogen, halogen, alkylether, amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl; Alkynyl, thio and alkylthio selected; and
R ₃ is a group of the formula -NR ₄ R ₅ in which
R _{4 is} a hydrogen atom or a group selected from alkyl, substituted alkyl or aryl-NH-C (Z) -, wherein Z is O, S or NR ^a , wherein R ^{a has} the meanings given above; and in case R _{4 is} hydrogen; Then it will be
R _{5 is selected} from R- and S-1-phenylethyl, benzyl, phenylethyl groups which are substituted or unsubstituted in one or more positions by a substituent selected from alkyl, amino, halogen, haloalkyl, nitro, hydroxyl, acetamido, Alkoxy and sulfonic acid or a salt thereof; Benzodioxanmethyl, fururyl, L-propylalanyl-aminobenzyl, β-alanylamino-benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or cycloalkyl; or R ₅ is a group of the following formula:

or, if R _{4 is} an alkyl or aryl-NH-C (Z) -, then R _{5 is} selected from the group consisting of heteroaryl-NR ^a -C (Z) -, heteroaryl-C (Z) -, alkaryl -NR ^a -C (Z) -, alkaryl-C (Z) -, aryl-NR-C (Z) - and aryl-C (Z) -; wherein Z is an oxygen, sulfur or amine; or a physiologically acceptable salt of the above-mentioned compound.

und physiologisch verträgliche Salze von besagter Verbindung, worin X₁, R₂ und R₄ wie oben definiert sind.More preferably, the A3RAg is a nucleoside derivative of the general formula (IV):

and physiologically acceptable salts of said compound wherein X ₁ , R ₂ and R _{4 are} as defined above.

The non-cyclic aliphatic groups (eg, alkyl, alkenyl, alkynyl, Alkoxy, aralkyl, alkaryl, alkylamine, etc.) which are part of the substituent form the compound of the present invention are either branched or linear aliphatic chains, preferably those containing one or more two to twelve Contain carbon atoms.

If on "physiological compatible Salts of the compounds, which are used by the present invention, reference is made If any non-toxic alkali metal, alkaline earth metal and ammonium salts, usually used in the pharmaceutical industry, including the Sodium, potassium, lithium, calcium, magnesium, barium, ammonium and protamine zinc salts by methods known in the art are to be produced. The term also includes non-toxic acid addition salts, generally by reacting the compounds of this invention prepared with a suitable organic or inorganic acid become. The acid addition salts are those that have the biological effectiveness and qualitative properties retained the free bases and non-toxic or otherwise undesirable are. Examples include inter alia acids derived from mineral acids are, hydrochloric acid, hydrobromic, Sulfuric acid, Nitric acid, Phosphoric acid, metaphosphoric and similar. Organic acids include inter alia tartaric acid, Acetic acid, propionic acid, Citric acid, maleic acid, malonic, Lactic acid, fumaric acid, benzoic acid, cinnamic acid, Mandelic, Glycolic acid, gluconic, Pyruvic acid, Succinic acid, salicylic acid and arylsulphonic acid, z. B. p-Toluolsulphonsäure.

Specific examples of A3RAg which can be used according to the general formula (IV) of the present invention include, but are not limited to, N ⁶ -2- (4-aminophenyl) ethyladenosine (APNEA), N ⁶ - (4-amino 3-iodobenzyl) adenosine 5 '- (N-methyluronamide) (AB-MECA) and N ⁶ - (2-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA) and preferably 2-chloro-N6- (2-iodobenzyl) adenosine 5'-N-methyluronamide (CI-IB-MECA).

Of Furthermore, the present invention provides the use of an A3RAg for the Making a drug available for the treatment of a disease or disease that is the activation of NK cells wherein said NK cells ex vivo through the contact activated by NK cells with an activating amount of A3RAg. According to one embodiment NK cells are autologous cells derived from the same individual were removed and then ex vivo by contacting this were activated with a lot of A3RAg to said activation to achieve, and then into the individual through a suitable parenteral Administration reintroduced were. Alternatively, you can the NK cells from a donor either after activation in vivo by administering the A3RAg to the donor for a sufficient period of time before removing the cells or by activating the cells in a culture as described above, or both. Method for the removal of relatively purified NK cell populations from an individual and their treatment in culture are on the Known and must therefore not further elaborated herein become.

Of the A3RAg can be formulated in several ways. He can as be formulated or in a pharmaceutically acceptable Salt to be converted. He can be alone or in combination with pharmaceutically acceptable Carriers, Diluents, Additives and adjuvants (generally referred to herein as pharmaceutical compatible Excipients).

If an A3RAg an individual for an in vivo treatment or an animal model for adoptive transfer treatment to disposal is preferably formulated for oral administration. however Other methods of administration are also suitable, such as parenteral Administration included the intravenous, subcutaneous, intramuscular, intramedullary injection, intra-arterial, intraperitoneal and intranasal administration, as well as intrathecal and infusion techniques. For oral administration is preferably A3RAg with good oral bioavailability selected. The search for an A3RAg with good oral bioavailability and good effect for achieving the desired therapeutic effect is for the expert a routine task.

If an A3RAg is administered orally, it is preferably for administration as a tablet, a suspension, a solution, an emulsion, a capsule, a powder, a syrup or similar formulated.

If If an A3RAg is administered parenterally, it will generally be administered in a unit dosage form for injection (solution, suspension, emulsion) be formulated and used as sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or Include dispersions.

It is mentioned, that people generally longer as experimental animals, as exemplified herein be treated as the treatment has a length that is proportional to the length of the disease process. The dosages can be single doses or Multiple doses over a period of, for. B. several days and can be from physical properties such as height, weight and gender depend on the individual to be treated. In general, the dosages administered, preferably unit dosage forms. The treatment generally has a length that coincides with the length and the stage of the disease process and the effectiveness of the active agent and the species to be treated.

Consequently The present invention also provides pharmaceutical compositions for the achievement of a therapeutic effect, wherein the composition pharmaceutically acceptable excipients and a Amount of an active ingredient that is effective to to achieve said therapeutic effect, wherein the therapeutic Effect includes the activation of NK cells and the active ingredient an A3RAg is. As experts in the field appreciate, For example, the composition of the invention may be a single A3RAg or contain a combination of two or more A3RAg.

Under the term "pharmaceutical compatible Auxiliaries "are meant any inert, non-toxic materials that are not react with A3RAg and which are typically used as formulations thinner or carrier or to shape or impart consistency to the formulation added to give it a specific form, e.g. In pill form, as a simple syrup, aromatic powder or other different To shape. The excipients can also be substances for imparting stability to the formulation (z. As preservatives) or to provide the formulation with an edible taste, etc.

The adjuvants may be any that are conventionally used and are limited only by chemical-physical considerations such as solubility and lack of reactivity with A3RAg and by the way of administration. The choice of excipient will be determined in part by the specific A3RAg used and by the particular method used to administer the composition. Accordingly, the excipient may include additives, dyes, diluents, buffers, disintegrants, moisturizers, preservatives, flavorants, and pharmacologically compatible carriers. In addition, the adjuvants may be an adjuvant, by definition, a substance that affects the action of the active ingredient in a predictable manner.

the accordingly pharmaceutical compositions for oral administration are suitable from (a) liquid solutions where there is an effective amount of A3RAg in diluents like water, saline, natural Juice, alcohols, syrup, etc. is dissolved; (b) capsules (e.g. the usual hard or soft coated gelatin type, the z. As surfactants, lubricants and inert filler contains) Tablets, lozenges (wherein the A3RAg is in a flavor such as sucrose and acacia or tragacanth, or the A3RAg is in an inert base such as gelatin and glycerin) and Pastilles, each containing a predetermined amount of A3RAg as solids or granules contain; (c) powder; (d) suspensions in a suitable liquid; (e) suitable emulsions; (f) liposome formulations; and other.

Sometimes may be an A3RAg in aerosol formulations for administration by Inhalation can be made. These aerosol formulations can be used in pressurized propellants such as dichlorodifluoromethane, Propane, nitrogen and the like. You can also as pharmaceuticals for Preparations without pressure, as in a nebuliser or atomizer, be formulated.

Of Further, the pharmaceutical compositions sometimes become formulated for parenteral administration and may include aqueous and non-aqueous, isotonic sterile injection solutions include antioxidants, buffers, bacteriostats and solutions contain the formulations isotonic with the blood of the intended receiver make and watery and not watery sterile suspensions, suspending agents, solubilizers, Thickeners, stabilizers and preservatives included. Oils like petroleum, animal, vegetable or synthetic oils and soaps such as fatty acid alkali metal, Ammonium and triethanolamine salts and suitable surfactants may also used for parenteral administration. Furthermore, in order to minimize or minimize irritation at the site of injection eliminate the compositions one or more non-ionic Containing surfactants. Suitable surfactants include polyethylene sorbitan fatty acid esters like sorbitan monooleate and the higher molecular weight adducts of ethylene oxide with a hydrophobic one Base produced by the condensation of propylene oxide with propylene glycol become.

The parenteral formulations may in sealed unit dose or multidose containers such as Ampoules and jars presents can and can stored in a freeze-dried (lyophilized) state only adding the sterile liquid carrier, e.g. As water, for injections immediately before use. Extremely porous injection solutions and Suspensions can from sterile powders, grains and tablets of the type described above.

The Invention will now be illustrated by way of example. It is to understand that these examples in their nature as an illustration are provided.

specific Examples

The effect of the synthetic A3 adenosine receptor agonist 2-chloro-N ⁶ - (2-iodobenzyl) -adenosine-5'-N-methyl-uronamide (CI-IB-MECA) on the in vitro, ex vivo and in vivo activity of NK cells tested.

Of the A3 adenosine receptor antagonist 9-chloro-2- (2-furanyl) -5 - [(phenylacetyl) amino] [1,2,4] triazolo [1,5-c] quinazoline (MRS-1220) was used to to demonstrate the specific binding of CI-IB-MECA to the A3AR.

All Drugs were purchased from RBI Massachusetts, USA. It becomes a stock solution by dissolving of 5 mg CI-IB-MECA or MRS1220 in dimethylsulfoxide (DMSO). Further dilutions were performed in RPMI.

murine Spleen cells were isolated from the spleen of ICR mice (Harlan Laboratories, Jerusalem, Israel) and human mononuclear cells were determined by a Ficoll hypaque gradient of heparinized Blood separated from healthy, normal donors.

example 1

The effect of CI-IB-MECA on the activity of human peripheral blood NK cells was examined by a standard 4 hour ⁵¹ Cr release assay using K562 leukemia cells as target cells. Human mononuclear cells were cultured at a concentration of 5 x 10 ⁵ cells / well in 96-well round-arch plates and used as effector (E) cells. The cells were preincubated with 10 nM CI-IB-MECA for 18 hours, then the agonist was washed out of the wells and the cells were resuspended in RPMI containing 10% FCS. K562 cells were used as the target cells (T) and were labeled with 100 μCi of Na ₂ [ ⁵¹ Cr] O ₄ at 37 ° C for 1 hour.

After extensive washing to remove the excess ⁵¹ Cr, the target cells (1 x 10 ⁴ ) were resuspended in RPMI and mixed with the effector cells in an E: T ratio of 1:50 in a total volume of 200 μl (triplicate assays). After 4 hours of incubation at 37 ° C in 5% CO ₂ , the plates were centrifuged and the supernatants were counted in a gamma counter (LKB).

The NK cytotoxicity was calculated using the following equation (CPM: counts per minute):

CPM spontaneous and CPM maximal were each determined by measuring the CPM (counts per minute) of the supernatants of the target cells in the presence of Na ₂ [ ⁵¹ Cr] O ₄ in the assay medium or in the presence of 1% Triton. It is noteworthy that the spontaneous release was below 10% of the maximum release over the duration of this experiment.

An increase in natural killer cell activity was observed after preincubation with CI-IB-MECA. The introduction of the A3 adenosine receptor antagonist MRS-1220 ended the stimulatory effect, which was the specific activation of the A3 adenosine receptor by CI-IB-MECA ( 1 ) shows.

Example 2

drugs

Cl-IB-MECA was dissolved in DMSO and then dilutions in PBS for received the in vivo experiments.

Ex vivo NK activity

Around to study the in vivo induction of NK cell activity by CI-IB-MECA was given to male ICR mice (Harlan Laboratories, Jerusalem, Israel), who were two months old and weighed on average 20 g, administered orally CI-IB-MECA.

Spleen cells prepared from the treated mice were removed for evaluation and NK cell activity was assayed by a standard 4 hour [ ⁵¹ Cr] release assay using YAC lymphoma cells as target cells. Specifically, spleen cells (1x10 ⁶ ) were cultured in 96 well round well plates and resuspended in RPMI containing 10% FBS. The YAC lymphoma cells were labeled with 100 μCi of Na ₂ [ ⁵¹ Cr] O ₄ at 37 ° C for 1 h. 2 x 10 ⁴ cells were then resuspended and mixed with the effector cells in the E: T ratio of 50: 1 in a volume of 200 μl. After 4 hrs of incubation, at 37 ° C in 5% CO ₂ , the plates were centrifuged and the supernatants were counted in a gamma counter (LKB). NK cytotoxicity was calculated using the equation described above.

Initially, the dose-dependent effect of CI-IB-MECA was determined by administering various dosages of the drug to mice (10-1000 μg / kg body weight) via the oral route. Spleen cells were separated from the mice 48h after treatment with the drug and tested for NK activity using the ⁵¹ Cr assay as described above. As in 2 Dose-dependent potentiation of NK activity by CI-IB-MECA was induced and maximum activity was observed at a dose of 10 μg / kg body weight.

In another experiment, the temporal kinetic effect of CI-IB-MECA (10 μg / kg body weight) on NK activity was investigated. In this experiment, mice were treated with the drug for four consecutive days. 4, 11 and 18 days after the last treatment, the mice were sacrificed and the spleen was removed. The spleen cells were separated and tested for NK activity using the ⁵¹ Cr assay as described above. In addition, serum samples were collected to assess IL-12 levels. Each group contained five mice and the study was repeated four times.

The results produced a bell-shaped response curve with maximum NK cell activity on day 4 after the last CI-IB-MECA treatment, reaching the control values on day 18 ( 3 ). An increase in the amount of cytokine IL-12 was observed in serum samples taken from CI-IB-MECA treated mice ( 4 ).

Example 3

tumor cells

B16-F10 Melanoma cells and HCT-116 human colon carcinoma cells were used in this set of experiments. The cells were in RPMI medium stored containing 10% fetal bovine serum (FBS). The cells were twice a week transferred to a freshly prepared medium.

In this in vivo study, C57B1 / 6J male mice (Harlan Laboratories, Jerusalem, Israel), two months old and weighing on average 25 g, were used as the artificial melanoma lung metastasis mouse model to develop metastatic lung foci 15 days after inoculation. The C57B1 / 6J mice were inoculated intravenously (iv) with B-16 melanoma cells (2.5 x 10 ⁵ ) and treated daily orally with 10 μg / kg body weight CI-IB-MECA (starting one day after tumor inoculation). As control served mice, which were treated only with the vehicle.

To 15 days were the mice sacrificed and the spleen was removed and serum samples were used for IL-12 quantitation collected. Each group contained 15 mice and the study was three Repeated times.

In yet another experiment, HCT-116 (1.2 x 10 ⁶ ) colon carcinoma cells were injected subcutaneously into the flank of nude / BalbC mice. The mice were orally treated daily with 10 μg / kg body weight CI-IB-MECA one day after tumor inoculation. As control served mice, which were treated only with carrier means. Tumor growth was assessed every 3-4 days beginning on day 13 post tumor inoculation by measuring the width (W) and length (L) of the tumor. Each group contained 10 mice and the study was repeated 3 times. Tumor size was calculated according to the following formula:

Additionally were Serum samples for collected an IL-12 quantification.

Significant inhibition was observed in the development of metastatic pulmonary foci and colon carcinoma size in CI-IB-MECA treated mice ( 5A . 5B ). The amount of IL-12 measured in the serum samples derived from CI-IB-MECA treated mice was in both tumor bearing models ( 5C and 5D ) 41% higher than the amount obtained with the control group. Increased NK cell activity was also demonstrated in spleen cells derived from CI-IB-MECA-treated melanoma or colon carcinoma-bearing mice compared to untreated mice ( 5E and 5F ).

Example 4

Adoptive transfer experiment

For an adoptive transfer experiment, spleen cells derived from B16-F10 melanoma-bearing mice treated with 10 μg / kg body weight CI-IB-MECA were designated as "activated" cells, and those from the control group were not reported as " activated "spleen cells. The ability of "activated" spleen cells to act against melanoma cells in vivo was investigated. "Non-activated" and "activated" spleen cells (see the definition above) were grafted onto mice that had been in the study four days previously B16-F10 melanoma cells (2.5 x 10 ⁵ ) were inoculated. The controls were the mice inoculated with the B16-F10 melanoma cells but not grafted with the spleen cells.

The Mice were sacrificed on day 15 and the lungs were removed. The number of Lung melanoma foci was scanned using a dissecting microscope counted. Each group contained ten mice and the study was done three times.

As in 6 For example, mice grafted with "activated" spleen cells showed marked inhibition in the development of metastatic pulmonary foci as compared to the number of foci that developed in mice grafted with "non-activated" spleen cells or with the control group. The results prove that CI-IB-MECA activates NK cells.

IL-12 analysis in serum samples

serum samples for the Analysis of IL-12 levels were obtained and by a commercial murine ELISA kit from R & D Systems, Minneapolis MN, examined.

statistical analysis

The Results were statistically using the Student's t-test assessed. For a statistical analysis was a comparison between the mean performed by different experiments. The criterion for statistical Significance was p <0.05.

discussion

The Results shown herein show that the A3AR agonist CI-IB-MECA induced IL-12 production followed by increased NK cell activity in vivo. Spleen cells from mice who were treated orally with CI-IB-MECA practiced one dose- and time-dependent increased NK activity when incubated ex vivo with target cells. The treatment of melanoma or colon carcinoma-bearing mice with CI-IB-MECA the production of IL-12, increased the NK cell activity and inhibited tumor development. In addition, spleen cells were out CI-IB-MECA treated Melanoma-bearing mice were derived on mice grafted with B16-F10 melanoma cells were able to develop the metastatic pulmonary foci to prevent.

Adenosine exercises different Effects on the immune system, including anti-inflammatory Activity, Modulation of cytokine production, inhibition of platelet aggregation, Induction of erythropoietin production, thus modulating the Lymphocyte function [Nilsson J. and Olsson AG, Atherosclerosis 53: 77-82, (1984); Ward AC, et al. Biochem. Biophys. Res. Commun. 224: 10-6 (1994); and Wilson NJ, Jaworowski A, Ward AC, Hamilton JA. cAMP enhances CSF-1-induced ERK Biochem. Biophys. Res. Commun. 244: 475-80 (1998)]. Adenosine was clinically linked to the immune system by observation related to SCID patients the enzyme adenosine deaminase lacks what they are able to do requires catabolizing adenosine [Giblet ER, et al. Lancet 2: 1067-9 (1972)]. Purine nucleotides have been reported to be multiple Effects on lymphoid cells [Priebe T. et al. Cell Immunol. 129: 321-8 (1990)]. Incubation of human NK cells with extracellular purine nucleotides resulted in a dose-dependent Inhibition of proliferation without effect on the capacity for lysis tumor target cells [Miller JS, et al. J. Immunol. 192: 7376-82 (1999)]. Dibutyryl cAMP or Forskulin (an agent used in cells) cAMP amount increased by activation of adenylyl cyclase) capable of inhibiting the cytolytic activity of NK cells across from exercise certain tumor target cells [Hall TJ., Et al. Clin. Exp. Immunol. 54: 493-500 (1983)]. The results, obtained with the adenosine receptor agonist show one Role for the A1 and A2 receptors in the regulation of murine NK cell activity. Priebe et al. (supra) reported that NK cell activity in mouse spleen lymphocytes Adinosin receptor A2 agonist was inhibited while potent A1 receptor agonists are more effective stimulators.

The Results shown herein show that the administration of a specific A3AR agonists in the activation of NK cells in vivo resulted.

IL-12 is a cytokine known to be a central inducer of Th1 responses and cell-mediated immunity by induction of NK cell activity and production of tumor necrosis factor [Trinchieri G. Annu. Rev. Immunol. 13: 151-76 (1995)]. Previous reports showed that adenosine analogues stimulate the production of IL-12 by activated human monocytes by stimulating A2A receptors and subsequent increase of cAMP inhibited [Link AA. et al. J. Immunol. 164: 436-42 (2000)]. Hasko et al. [Hasko G. et al. FASEB 14: 2065-74 (2000)] demonstrated that adenosine receptor agonists inhibited the production of IL-12 in peritoneal MQ in the following order of potency GCS-21680 (A2AR agonist)> IB-MECA (A3AR agonist)> CCPA (A1AR Agonists). These results suggest that A2 adenosine receptors have a dominant role in inhibiting IL-12 production by adenosine agonists. In addition, it was shown that the effect of IB-MECA was biphasic, ie the inhibition of IL-12 production reached its maximum at 10 μm, while at 100 μm IB-MECA increased the production of IL-12 [Hasko G. et al , supra]. On the other hand, the same group of Hasko & Szabo showed that the IP administration of IB-MECA (500 μg / kg) to mice as a pretreatment to a lethal dose of an endotoxin, polypolysaccharide, improved the survival rate of the mice. LPS treatment caused the advent of large amounts of IL-12 in the plasma, while IB-MECA pretreatment suppressed the levels of IL-12 in the plasma. It should be noted that this effect of IB-MECA was only visible when animals were pretreated with the drug and was observed until 8 hours after treatment [Hasko G. et al. Eur. J. Pharmacol. 358: 261-8 (1998)]. In this study, administration of low doses of CI-IB-MECA induced IL-12 production, followed by induction of NK activity. Thus, it can be concluded that an A3AR agonist induces IL-12 production, thereby stimulating NK activity, which plays a role in CI-IB-MECA antitumor action.

The Results shown herein show that oral administration increased levels of low doses of an A3AR agonist (CI-IB-MECA) induced by serum IL-12, resulting in increased NK cell activity and tumor growth inhibition was followed. These results show that chronic activation of the receptor in an opposite effect, compared to such which are observed with acute activation of the receptor result could.

Claims (12)

Use of A3RAg (A3 receptor agonist) for Preparation of a pharmaceutical composition for treatment an infectious Illness. Use according to claim 1, being the infectious Disease caused by an agent selected from: a virus, a bacterium and a protozoan. The use according to claim 1, wherein said A3RAg is a compound of the general formula (I):

where R _{1 represents} an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or a group of the following general formula (II):

in which: Y represents oxygen, sulfur or CH ₂ ; - X ₁ is H, alkyl, R ^a R ^b NC (= O) - or HOR ^c - represents, wherein - R ^a and R ^b may be identical or different and are selected from hydrogen, alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl, or joined together to form a heterocyclic ring containing from two to five carbon atoms; and R ^{c is} selected from alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl; X _{2 is} H, hydroxyl, alkylamino, alkylamido or hydroxyalkyl; - X ₃ and X _{4 are} independently hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, -OCOPh, -OC (= S ) OPh or on both sides of X ₃ and X _{4 are} oxygen, which are connected to> C = S so that they form a 5-membered ring, or X ₂ and X _{3 form} a ring of formula (III):

wherein R 'and R "independently represent an alkyl group; R _{2 is} selected from hydrogen, halo, alkyl ethers, amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl; Alkynyl, thio and alkylthio; and R _{3 is} a group of the formula -NR ₄ R ₅ , wherein - R _{4 is} a hydrogen atom or a group selected from alkyl, substituted alkyl or aryl-NH-C (Z) -, where Z, O, S, or NR ^a , wherein R ^{a has} the meanings described above; and in case R _{4 is} hydrogen; R _{5 is} selected from R- and S-1-phenylethyl, benzyl, phenylethyl groups, which are unsubstituted or substituted in one or more positions with a substituent selected from alkyl, amino, halo, haloalkyl, nitro, hydroxyl, Acetoamido, alkoxy and sulfonic acid or a salt thereof; Benzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl, β-alanylaminobenzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or cycloalkyl; or R ₅ is a group of the following formula:

or when R _{4 is} an alkyl or aryl-NH-C (Z) -, R _{5 is} selected from heteroaryl-NR ^a -C (Z) -, heteroaryl-C (Z) -, alkylaryl-NR ^a -C ( Z) -, alkaryl-C (Z) -, aryl-NR-C (Z) - and aryl-C (Z) -; Z represents oxygen, sulfur or amine; or a pharmaceutically acceptable salt of the above-mentioned compound. The use according to claim 3, wherein A3RAg is a nucleoside derivative of the general formula (IV):

wherein X ₁ , R ₂ and R _{4 are} as defined in claim 3. The use according to claim 1, wherein said A3RAg is selected from N ⁶ -2- (4-aminophenyl) ethyladenosine (APNEA), N ⁶ - (4-amino-3-iodobenzyl) adenosine-5 '- (N-methyl Uronamide) (AB-MECA), N ⁶ - (3-iodobenzyl) adenosine-5'-N-methyl-ureamide (IB-MECA), and 2-chloro-N ⁶ - (3-iodobenzyl) adenosine-5 'N-Methyl-Uronamide (CI-IB-MECA). The use according to claim 5, wherein said A3RAg C1-IB-MECA is. The use according to any one of claims 1 to 6 for the preparation of a pharmaceutical composition intended for oral Administration is suitable. A method of activating natural killer (NK) cells, which comprises contacting said cells ex vivo with a such amount of agent that is responsible for the activation of said NK cells is effective, said active agent being an adenosine A3 receptor agonist (A3RAg) is. The method of claim 8, wherein said A3RAg is a compound of the general formula (I):

where R _{1 represents} an alkyl, hydroxyalkyl, carboxyalkyl or cyanoalkyl or a group of the following general formula (II):

in which: Y represents an oxygen, sulfur or CH ₂ ; - X ₁ is H, alkyl, R ^a R ^b NC (= O) - or HOR ^c - represents, in which - R ⁸ and R ^b may be the same or different and are selected from hydrogen, alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl and cycloalkyl, or may be linked together to form a heterocyclic ring containing from two to five carbon atoms; and R ^{c is} selected from alkyl, amino, haloalkyl, aminoalkyl, BOC-aminoalkyl, and cycloalkyl; X _{2 is} H, hydroxyl, alkylamino, alkylamido or hydroxyalkyl; - X ₃ and X _{4 are} independently hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, -OCOPh, -OC (= S ) OPh represent, or on both sides X ₃ and X ₄ are oxygens which are linked to> C = S to form a five-membered ring, or X ₂ and X ₃ (form III) the ring of the formula:

wherein R 'and R "independently represent an alkyl group; R _{2 is} selected from hydrogen, halo, alkyl ethers, amino, hydrazido, alkylamino, alkoxy, thioalkoxy, pyridylthio, alkenyl, alkynyl, thio and alkylthio; and - R _{3 is} a group of the formula -NR ₄ R ₅ , wherein - R _{4 is} a hydrogen atom or a group selected from alkyl, substituted alkyl or aryl-NH-C (Z) -, wherein Z is an O, S or NR ^a is wherein R ^{a has} the meanings given above; wherein in the case where R _{4 is} hydrogen, R _{5 is} selected from R- and S-1-phenylethyl, benzyl, phenylethyl groups which are unsubstituted or substituted at one or more positions with a substituent selected from alkyl, Amino, halo, haloalkyl, nitro, hydroxyl, acetoamido, alkoxy, and sulfonic acid, or a salt thereof; Benzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl, β-alanylaminobenzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or cycloalkyl; or R ₅ is a group of the following formula:

or when R _{4 is} an alkyl or aryl-NH-C (Z) -, R _{5 is} selected from the group consisting of heteroaryl-NR ^a -C (Z) -, heteroaryl-C (Z) -, alkaryl-NR ^a -C (Z) -, alkaryl-C (Z) -, aryl-NR-C (Z) - and aryl-C (Z) -; Z represents oxygen, sulfur or amine; or a pharmaceutically acceptable salt of the above-mentioned compound. The method of claim 9, wherein said A3RAg is a compound of the general formula (IV):

wherein X ₁ , R ₂ and R _{4 are} as defined in the claim. The method of claim 10, wherein A3RAg is selected from N ⁶ -2- (4-aminophenyl) ethyladenosine (APNEA), N ⁶ - (4-amino-3-iodobenzyl) adenosine-5 '- (N-methyl uronamide ) (AB-MECA), N ⁶ - (3-iodobenzyl) -adenosine-5'-N-methyl-ureamide (IB-MECA), and 2-chloro-N ⁶ - (3-iodobenzyl) adenosine-5'N -Methyl uronamide (C1-IB-MECA). The method of claim 11, wherein said A3RAg C1-IB-MECA is.